Hyaluronic acid is a major component of the extracellular matrix that surrounds migrating and proliferating cells. It is an extremely long, negatively charged polysaccharide, each molecule of which consists of as many as 50,000 replications of the simple disaccharide composed by Glucuronic acid and N-Acetylglucosamine. Indeed, it is important to mention successful in vivo applications of its derivatives in wound healing, diabetic ulcers, vitiligo treatments, laparoscopic adhesion prevention, anti-adhesive surface coatings, cartilage regeneration.
One class of HA derivatives are called auto-crosslinked polysaccharides. In this case the stability if the polymer was obtained by creating cross-linking bonds, but no additional chemicals were involved in bridging the hyaluronan chains. Stabilization was achieved by directly esterifying a certain percentage of the carboxyl groups of glucuronic acid along the polymeric chain with hydroxyl groups of the same or different hyaluronan molecules. These materials are highly viscous suspensions in sterile distilled water of auto-reticulate microparticles with variable concentrations of between 30 and 60 mg/mL.
An alternative way of obtaining HA derivatives involves the use of coupling reactions where specific functional groups of the polysaccharide (e.g. carboxyl, hydroxyl, N-acetyl groups) are modified by chemical reactions such as esterification, sulphatation, amidation and so on.
All of these HA modified biomaterials in vivo spontaneously degrade by enzymatic activity or hydrolysis, giving fragments of HA molecules and the functional groups previously bound (benzyl or ethyl alcohol, amides, etc.)
The synthesis of HA derivatives usually involves a two-step procedure: the preparation of a quaternary salt of HA and its subsequent reaction with a chemical agent (esterifying, amidating, etc) in aprotic solvent at a controlled temperature. A variety of alcohols can be used for esterification (aliphatic, araliphatic, cycloaliphatic and others). Amidated HA can be prepared according to EP 1095064. Hyaluronic acid esters are known for instance from EP 216453. O-sulphated derivatives of HA are known from EP 702699.
Percarboxylated derivatives of HA are known from EP 1339753.
According to the chemical group substituted during chemical reactions (and the percentage of substitution too) the resulting biomaterials could provide totally different mechanical properties: for example, in the case of esterified HA, the residence time increases by means of increased hydrophobicity and decreased negative charge of the carboxyl group.
Keratinocytes, fibroblasts, chondrocytes, mesenchymal stem cells, endothelial cells, hepatocytes, urethelial cells and nerve cells have proven to proliferate efficiently on HA modified biomaterials. However, the vast majority of the cross-linked HA derivatives previously mentioned are represented by very hydrated materials, which are not cell-adhesive and, for this reason, were initially considered as resorbable materials for the prevention of surgical adhesions.
Our first tissue engineering approach was to test this important variety of fully biodegradable biomaterials. HA-based products have received European Community approval for clinical applications, and three products have been FDA-approved.
SCI (Spinal Cord Injury) may be characterized as a result of continuing processes of tissue destruction, abortive repair, and wound healing around the injury site. A significant body of evidence suggests that SCI evolves through three phases: the acute, secondary, and chronic phase. The initial core lesion progressively expands and the SCI evolves into its chronic phase. White matter demonstrates partial or complete demyelinization that is responsible for conduction deficits. Approximately 25% of SCI patients develop a centrally located cyst that progressively expands leading to syringomyelia.
Further histopathologic features include gray matter dissolution, connective tissue deposition and gliosis. The range and location of injury determine the overall neurological deficit, development of hyperexcitability, and chronic pain syndromes.
If the main area of research interest is the physiologic response to injury or the pathophysiology of secondary injury, then the investigator might choose a contusion model, because it closely parallels human SCI. However, the unambiguous demonstration of regenerating axons in a contusion model poses a new challenge, specifically the delineation of spared and regenerated axons.
Behavioral studies and statistical evaluation of motor-sensory pathway recovery too could be an extremely critical step with contusion models, into which animal control groups (injured but not treated) spontaneously recover part of their lost nervous connectivity.
If a device is to be implemented, a partial or complete transection model might be best suited for device placement. For certain experimental paradigms, a combination of models might be planned. For example, the early stages of an experimental plan that explores axon regeneration might utilize transection models to demonstrate unambiguously regenerated axons and identify the most promising therapies, which can then be tested in contusion models by analysis techniques.
In a transection model the ability to differentiate spared axons from regenerated ones is not an issue. For certain applications, unilateral hemisection injury can be a viable alternative to complete transection. A major advantage of this approach is the preserved structural integrity and function of one side of the spinal cord. Unilateral spinal cord sparing is usually sufficient to maintain bladder and bowel function, which results in less-intensive post-operative animal care.
For these reasons, spinal cord hemisection in rats was used as an experimental animal model for testing spinal cord regeneration after injury and implantation of a scaffold made of esterified HA whereas, for testing peripheral nerve regeneration, a complete transection of the sciatic nerve has been carried out.